Pursuant to 37 C.F.R. 1.71 (e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
Recursive sequence recombination entails performing iterative cycles of recombination and screening or selection to xe2x80x9cevolvexe2x80x9d individual genes, whole plasmids or viruses, multigene clusters, or even whole genomes (Stemmer, Bio/Technology 13:549-553 (1995)). Such techniques do not require the extensive analysis and computation required by conventional methods for polypeptide engineering. Recursive sequence recombination allows the recombination of large numbers of mutations in a minimum number of selection cycles, in contrast to traditional, pairwise recombination events.
Thus, recursive sequence recombination (RSR) techniques provide particular advantages in that they provide recombination between mutations in any or all of these, thereby providing a very fast way of exploring the manner in which different combinations of mutations can affect a desired result.
In some instances, however, structural and/or functional information is available which, although not required for recursive sequence recombination, provides opportunities for modification of the technique. In other instances, selection and/or screening of a large number of recombinants can be costly or time-consuming. A further problem can be the manipulation of large nucleic acid molecules. The instant invention addresses these issues and others.
One aspect of the invention is a method for evolving a protein encoded by a DNA substrate molecule comprising:
(a) digesting at least a first and second DNA substrate molecule, wherein the at least a first and second substrate molecules differ from each other in at least one nucleotide, with a restriction endonuclease;
(b) ligating the mixture to generate a library of recombinant DNA molecules;
(c) screening or selecting the products of (b) for a desired property; and
(d) recovering a recombinant DNA substrate molecule encoding an evolved protein.
A further aspect of the invention is a method for evolving a protein encoded by a DNA substrate molecule by recombining at least a first and second DNA substrate molecule, wherein the at least a first and second substrate molecules differ from each other in at least one nucleotide and comprise defined segments, the method comprising:
(a) providing a set of oligonucleotide PCR primers, comprising at least one primer for each segment, wherein the primer sequence is complementary to at least one junction with another segment;
(b) amplifying the segments of the at least a first and second DNA substrate molecules with the primers of step (a) in a polymerase chain reaction;
(c) assembling the products of step (b) to generate a library of recombinant DNA substrate molecules;
(d) screening or selecting the products of (c) for a desired property; and
(e) recovering a recombinant DNA substrate molecule from (d) encoding an evolved protein.
A further aspect of the invention is a method of enriching a population of DNA fragments for mutant sequences comprising:
(a) denaturing and renaturing the population of fragments to generate a population of hybrid double-stranded fragments in which at least one double-stranded fragment comprises at least one base pair mismatch;
(b) fragmenting the products of (a) into fragments of about 20-100 bp;
(c) affinity-purifying fragments having a mismatch on an affinity matrix to generate a pool of DNA fragments enriched for mutant sequences; and
(d) assembling the products of (c) to generate a library of recombinant DNA substrate molecules.
A further aspect of the invention is a method for evolving a protein encoded by a DNA substrate molecule, by recombining at least a first and second DNA substrate molecule, wherein the at least a first and second substrate molecules share a region of sequence homology of about 10 to 100 base pairs and comprise defined segments, the method comprising:
(a) providing regions of homology in the at least a first and second DNA substrate molecules by inserting an intron sequence between at least two defined segments;
(b) fragmenting and recombining DNA substrate molecules of (a), wherein regions of homology are provided by the introns;
(c) screening or selecting the products of (b) for a desired property; and
(d) recovering a recombinant DNA substrate molecule from the products of (c) encoding an evolved protein.
A further aspect of the invention is a method for evolving a protein encoded by a DNA substrate molecule by recombining at least a first and second DNA substrate molecule, wherein the at least a first and second substrate molecules differ from each other in at least one nucleotide and comprise defined segments, the method comprising:
(a) providing a set of oligonucleotide PCR primers, wherein for each strand of each segment a pair of primers is provided, one member of each pair bridging the junction at one end of the segment and the other bridging the junction at the other end of the segment, with the terminal ends of the DNA molecule having as one member of the pair a generic primer, and wherein a set of primers is provided for each of the at least a first and second substrate molecules;
(b) amplifying the segments of the at least a first and second DNA substrate molecules with the primers of (a) in a polymerase chain reaction;
(c) assembling the products of (b) to generate a pool of recombinant DNA molecules;
(d) selecting or screening the products of (c) for a desired property; and
(e) recovering a recombinant DNA substrate molecule from the products of (d) encoding an evolved protein.
A further aspect of the invention is a method for optimizing expression of a protein by evolving the protein, wherein the protein is encoded by a DNA substrate molecule, comprising:
(a) providing a set of oligonucleotides, wherein each oligonucleotide comprises at least two regions complementary to the DNA molecule and at least one degenerate region, each degenerate region encoding a region of an amino acid sequence of the protein;
(b) assembling the set of oligonucleotides into a library of full length genes;
(c) expressing the products of (b) in a host cell;
(d) screening the products of (c) for improved expression of the protein; and
(e) recovering a recombinant DNA substrate molecule encoding an evolved protein from (d).
A further aspect of the invention is a method for optimizing expression of a protein encoded by a DNA substrate molecule by evolving the protein, wherein the DNA substrate molecule comprises at least one lac operator and a fusion of a DNA sequence encoding the protein with a DNA sequence encoding a lac headpiece dimer, the method comprising:
(a) transforming a host cell with a library of mutagenized DNA substrate molecules;
(b) inducing expression of the protein encoded by the library of (a);
(c) preparing an extract of the product of (b);
(d) fractionating insoluble protein from complexes of soluble protein and DNA; and
(e) recovering a DNA substrate molecule encoding an evolved protein from (d).
A further aspect of the invention is a method for evolving functional expression of a protein encoded by a DNA substrate molecule comprising a fusion of a DNA sequence encoding the protein with a DNA sequence encoding filamentous phage protein to generate a fusion protein, the method comprising:
(a) providing a host cell producing infectious particles expressing a fusion protein encoded by a library of mutagenized DNA substrate molecules;
(b) recovering from (a) infectious particles displaying the fusion protein;
(c) affinity purifying particles displaying the mutant protein using a ligand for the protein; and
(d) recovering a DNA substrate molecule encoding an evolved protein from affinity purified particles of (c).
A further aspect of the invention is a method for optimizing expression of a protein encoded by a DNA substrate molecule comprising a fusion of a DNA sequence encoding the protein with a lac headpiece dimer, wherein the DNA substrate molecule is present on a first plasmid vector, the method comprising:
(a) providing a host cell transformed with the first vector and a second vector comprising a library of mutants of at least one chaperonin gene, and at least one lac operator;
(b) preparing an extract of the product of (a);
(c) fractionating insoluble protein from complexes of soluble protein and DNA; and
(d) recovering DNA encoding a chaperonin gene from (c).
A further aspect of the invention is a method for optimizing expression of a protein encoded by a DNA substrate molecule comprising a fusion of a DNA sequence encoding the protein with a filamentous phage gene, wherein the fusion is carried on a phagemid comprising a library of chaperonin gene mutants, the method comprising:
(a) providing a host cell producing infectious particles expressing a fusion protein encoded by a library of mutagenized DNA substrate molecules;
(b) recovering from (a) infectious particles displaying the fusion protein;
(c) affinity purifying particles displaying the protein using a ligand for the protein; and
(d) recovering DNA encoding the mutant chaperonin from affinity purified particles of (c).
A further aspect of the invention is a method for optimizing secretion of a protein in a host by evolving a gene encoding a secretory function, comprising:
(a) providing a cluster of genes encoding secretory functions;
(b) recombining at least a first and second sequence in the gene cluster of (a) encoding a secretory function, the at least a first and second sequences differing from each other in at least one nucleotide, to generate a library of recombinant sequences;
(c) transforming a host cell culture with the products of (b), wherein the host cell comprises a DNA sequence encoding the protein;
(d) subjecting the product of (c) to screening or selection for secretion of the protein; and
(e) recovering DNA encoding an evolved gene encoding a secretory function from the product of (d).
A further aspect of the invention is a method for evolving an improved DNA polymerase comprising:
(a) providing a library of mutant DNA substrate molecules encoding mutant DNA polymerase;
(b) screening extracts of cells transfected with (a) and comparing activity with wild type DNA polymerase;
(c) recovering mutant DNA substrate molecules from cells in (b) expressing mutant DNA polymerase having improved activity over wild-type DNA polymerase; and
(d) recovering a DNA substrate molecule encoding an evolved polymerase from the products of (c).
A further aspect of the invention is a method for evolving a DNA polymerase with an error rate greater than that of wild type DNA polymerase comprising:
(a) providing a library of mutant DNA substrate molecules encoding mutant DNA polymerase in a host cell comprising an indicator gene having a revertible mutation, wherein the indicator gene is replicated by the mutant DNA polymerase;
(b) screening the products of (a) for revertants of the indicator gene;
(c) recovering mutant DNA substrate molecules from revertants; and
(d) recovering a DNA substrate molecule encoding an evolved polymerase from the products of (c).
A further aspect of the invention is a method for evolving a DNA polymerase, comprising:
(a) providing a library of mutant DNA substrate molecules encoding mutant DNA polymerase, the library comprising a plasmid vector;
(b) preparing plasmid preparations and extracts of host cells transfected with the products of (a);
(c) amplifying each plasmid preparation in a PCR reaction using the mutant polymerase encoded by that plasmid, the polymerase being present in the host cell extract;
(d) recovering the PCR products of (c); and
(e) recovering a DNA substrate molecule encoding an evolved polymerase from the products of (d).
A further aspect of the invention is a method for evolving a p-nitrophenol phosphonatase from a phosphonatase encoded by a DNA substrate molecule, comprising:
(a) providing library of mutants of the DNA substrate molecule, the library comprising a plasmid expression vector;
(b) transfecting a host, wherein the host phn operon is deleted;
(c) selecting for growth of the transfectants of (b) using a p-nitrophenol phosphonatase as a substrate;
(d) recovering the DNA substrate molecules from transfectants selected from (c); and
(e) recovering a DNA substrate molecule from (d) encoding an evolved phosphonatase.
A further aspect of the invention is a method for evolving a protease encoded by a DNA substrate molecule comprising:
(a) providing library of mutants of the DNA substrate molecule, the library comprising a plasmid expression vector, wherein the DNA substrate molecule is linked to a secretory leader;
(b) transfecting a host;
(c) selecting for growth of the transfectants of (b) on a complex protein medium; and
(d) recovering a DNA substrate molecule from (c) encoding an evolved protease.
A further aspect of the invention is a method for screening a library of protease mutants displayed on a phage to obtain an improved protease, wherein a DNA substrate molecule encoding the protease is fused to DNA encoding a filamentous phage protein to generate a fusion protein, comprising:
(a) providing host cells expressing the fusion protein;
(b) overlaying host cells with a protein net to entrap the phage;
(c) washing the product of (b) to recover phage liberated by digestion of the protein net;
(d) recovering DNA from the product of (c); and
(e) recovering a DNA substrate from (d) encoding an improved protease.
A further aspect of the invention is a method for screening a library of protease mutants to obtain an improved protease, the method comprising:
(a) providing a library of peptide substrates, the peptide substrate comprising a fluorophore and a fluorescence quencher;
(b) screening the library of protease mutants for ability to cleave the peptide substrates, wherein fluorescence is measured; and
(c) recovering DNA encoding at least one protease mutant from (b).
A further aspect of the invention is a method for evolving an alpha interferon gene comprising:
(a) providing a library of mutant alpha interferon genes, the library comprising a filamentous phage vector;
(b) stimulating cells comprising a reporter construct, the reporter construct comprising a reporter gene under control of an interferon responsive promoter, and wherein the reporter gene is GFP;
(c) separating the cells expressing GFP by FACS;
(d) recovering phage from the product of (c); and
(e) recovering an evolved interferon gene from the product of (d).
A further aspect of the invention is a method for screening a library of mutants of a DNA substrate encoding a protein for an evolved DNA substrate, comprising:
(a) providing a library of mutants, the library comprising an expression vector;
(b) transfecting a mammalian host cell with the library of (a), wherein mutant protein is expressed on the surface of the cell;
(c) screening or selecting the products of (b) with a ligand for the protein;
(d) recovering DNA encoding mutant protein from the products of (c); and
(e) recovering an evolved DNA substrate from the products of (d).
A further aspect of the invention is a method for evolving a DNA substrate molecule encoding an interferon alpha, comprising:
(a) providing a library of mutant alpha interferon genes, the library comprising an expression vector wherein the alpha interferon genes are expressed under the control of an inducible promoter;
(b) transfecting host cells with the library of (a);
(c) contacting the product of (b) with a virus;
(d) recovering DNA encoding a mutant alpha interferon from host cells surviving step (c); and
(e) recovering an evolved interferon gene from the product of (d).
A further aspect of the invention is a method for evolving the stability of a protein encoded by a DNA substrate molecule, the DNA substrate molecule comprising a fusion of a DNA sequence encoding the protein with a DNA sequence encoding a filamentous phage protein to generate a fusion protein, the method comprising:
(a) providing a host cell expressing a library of mutants of the fusion protein;
(b) affinity purifying the mutants with a ligand for the protein, wherein the ligand is a human serum protein, tissue specific protein, or receptor;
(c) recovering DNA encoding a mutant protein from the affinity selected mutants of (b); and
(d) recovering an evolved gene encoding the protein from the product of (c).
A further aspect of the invention is a method for evolving a protein having at least two subunits, comprising:
(a) providing a library of mutant DNA substrate molecules for each subunit;
(b) recombining the libraries into a library of single chain constructs of the protein, the single chain construct comprising a DNA substrate molecule encoding each subunit sequence, the subunit sequence being linked by a linker at a nucleic acid sequence encoding the amino terminus of one subunit to a nucleic acid sequence encoding the carboxy terminus of a second subunit;
(c) screening or selecting the products of (B),
(d) recovering recombinant single chain construct DNA substrate molecules from the products of (c);
(e) subjecting the products of (d) to mutagenesis; and
(f) recovering an evolved single chain construct DNA substrate molecule from (e).
A further aspect of the invention is a method for evolving the coupling of a mammalian 7-transmembrane receptor to a yeast signal transduction pathway, comprising:
(a) expressing a library of mammalian G alpha protein mutants in a host cell, wherein the host cell expresses the mammalian 7-transmembrane receptor and a reporter gene, the receptor gene geing expressed under control of a pheromone responsive promoter;
(b) screening or selecting the products of (a) for expression of the reporter gene in the presence of a ligand for the 7-transmembrance receptor; and
(c) recovering DNA encoding an evolved G alpha protein mutant from screened or selected products of (b).
A further aspect of the invention is a method for recombining at least a first and second DNA substrate molecule, comprising:
(a) transfecting a host cell with at least a first and second DNA substrate molecule wherein the at least a first and second DNA substrate molecules are recombined in the host cell;
(b) screening or selecting the products of (a) for a desired property; and
(c) recovering recombinant DNA substrate molecules from (b).
A further aspect of the invention is a method for evolving a DNA substrate sequence encoding a protein of interest, wherein the DNA substrate comprises a vector, the vector comprising single-stranded DNA, the method comprising:
(a) providing single-stranded vector DNA and a library of mutants of the DNA substrate sequence;
(b) annealing single stranded DNA from the library of (a) to the single stranded vector DNA of (a);
(c) transforming the products of (b) into a host;
(d) screening the product of (c) for a desired property; and
(e) recovering evolved DNA substrate DNA from the products of (d).